Molecular Epidemiology of Pulmonary Tuberculosis in Southern Ethiopia

Understanding the epidemiology of tuberculosis (TB) is limited by lack of genotyping data. We sought to characterize the drug susceptibility testing (DST) patterns and genetic diversity of M. tuberculosis (Mtb) isolates in Southern Ethiopia. A cross-sectional study was conducted among newly diagnosed sputum smear positive patients with TB visiting nine health facilities in southern Ethiopia from June 2015 to May 2016. Three consecutive sputum samples (spot-morning-spot) per patient were examined using acid-fast bacilli (AFB) smear microscopy with all smear positive specimens having AFB cultures performed. Mtb isolates had DST performed using indirect proportion method and were genotyped with RD9 deletion typing and spoligotyping. Spoligotyping International Types (SIT) and sub-lineages (clades) were assigned according to the SITVITWEB data base. The study identified a heterogeneous pool of M. tuberculosis strains with several clusters including lineage 7 strains circulating in southern Ethiopia. No MDR-TB case was detected in the study area; however, the high proportion of INH monoresistance is a significant risk for the potential emergence of MDR-TB in the study area as INH monoresistance is the initial step towards anti-TB drug resistance and a common pathway to the development of MDR-TB. SIT 149 (T3-ETH) was the most dominant strain cluster circulating in the study area, including among drug resistant cases. The findings highlight the ongoing transmission of TB, including of drug resistant TB, in southern Ethiopia and call for surveillance and wider monitoring of DST, supported by geospatial analysis to monitor transmission trends and improve control responses.

The study revealed several clusters including lineage 7 strains circulating in southern Ethiopia. SIT 149 (T3-ETH) was the most dominant circulating strain in the study area including among drug-resistant cases. Background Tuberculosis (TB) remains a major and urgent global public health problem, especially in low-income countries where the burden of disease is high. Globally, there were 10 million new cases and 1.5 million deaths among persons with TB in 2017 (1). The spread of TB is fueled by several factors including the HIV epidemic, low socioeconomic status, overcrowding, malnutrition, and the emergence of drug-resistance (2)(3)(4).
Drug-resistant TB is a major public health concern in many countries and continues to be a public health crisis (1). Ethiopia is one of fourteen countries appearing in all three WHO high burden country lists for TB, TB/HIV and MDR-TB. According to the national antituberculosis drug resistance survey in 2014, the prevalence of MDR-TB among new and previously treated TB cases was 2.3% and 17.8% respectively (5). WHO estimated the prevalence of MDR-TB among new TB cases in Ethiopia at 2.7% and among previously treated cases at 14% in 2017 (1). Additionally, data from different parts of the country show that drug-resistant TB is a major public health concern that demands attention (6)(7)(8).
Interruption of the transmission of M. tuberculosis is one of the primary goals of tuberculosis control programs. Tracking specific strains of M.tuberculosis circulating in the community informs public health authorities on patterns of spread and potential areas for action to curb the spread of TB in communities (9). Spoligotyping is a PCR-based method commonly used to characterize of M. tuberculosis strains circulating in a community (10) and strain differentiation is based on the polymorphism in the direct repeat (DR) locus, which is a distinct chromosomal region in Mtb genome (11).
While several molecular epidemiological studies have been conducted to describe the diversity and drug susceptibility profile of M. tuberculosis strains in various geographical areas of Ethiopia (7,(12)(13)(14), data from the south, where population density is relatively higher, is limited. The goal of our study was to characterize the drug susceptibility patterns and genetic diversity of M. tuberculosis isolates circulating in southern Ethiopia.

Study setting/design
This cross-sectional study was conducted at nine health facilities (two hospitals and seven health centers) in and around Shashemene area, in West Arisi Zone of Oromia Region, Ethiopia. Shashemene is a major urban center and commercial town, located 240 km south of the capital Addis Ababa. The estimated population of the study area including of Shashemene and the adjacent rural towns of Wondo Genet, Aje, and Arsi-Negele was 3 million people. All nine health facilities included in the study area provided services for the diagnosis and treatment of TB through DOTS clinics. Patients enrolled at the nine health facilities from the West Arsi Zone and adjoining kebele of Wondo Genet were mapped and fell into seven districts (Woredas) (Fig. 1).

Study population and variables
Among the persons suspected to have TB who were investigated at any one of the nine health facilities during the study period, all newly diagnosed sputum smear positive pulmonary TB patients who provided written informed consent for study participation were enrolled. Assent for the children and consent by parents or guardians for those under 18 yrs of age were also obtained. The study was conducted from June 2015 through May 2016. Three consecutive sputum samples (spot-morning-spot) were collected from each TB suspect. Socio-demographic and clinical information was obtained for all study participants by a TB clinic nurse at the respective health facilities using a pre-tested standard questionnaire. Data on HIV status was retrieved from health facility records.

Specimen Collection
Sputum smears were prepared and examined by an onsite health facility laboratory technologist. The remaining portion of the three sputum samples from AFB smear positive patients were pooled individually into 50 ml sterile screw capped universal test tubes and stored at the diagnostic centers at -20˚C for a maximum of one week until transported on cold chain to the Armauer Hansen Research Institute (AHRI) in Addis Ababa, Ethiopia for mycobacterial culture and molecular testing.

Mycobacterial Culture
The pooled sputum samples were processed at AHRI within one day based on standard procedures as previously described (15). In brief, the sputum samples were digested and decontaminated using Petroff's method and the processed sample was inoculated into three tubes containing egg-based Löwenstein Jensen (LJ) media (two with glycerol and one with pyruvate). The inoculated media were incubated at 37 o C for at least 8 weeks, with weekly observation for the presence of mycobacterial colonies. Cultures with no growth after the eighth week were considered negative. Mycobacterial growth was confirmed by typical colony morphology and AFB staining.

Drug Susceptibility Test (DST)
The conventional indirect proportion method was employed to perform DST using 7H10 medium on 24-well tissue culture plates using a standard protocol (16). In brief, four first line anti-TB drugs (isoniazid, rifampicin, ethambutol and streptomycin) were mixed with the agar media at recommended concentration and dispensed into 9 wells of the 24 well tissue culture plates and two wells were dispensed with drug free media. The agar plate was sealed with parafilm and incubated in an inverted position at 35˚C. The plates were checked on day 6, 12 and 19 for evidence of growth. Resistance was expressed as the percentage of colonies that grew on critical concentrations of the drugs, i.e. 0.2 µg/ml for isoniazid (INH), 1 µg/ml for rifampicin (RPM), 5 µg/ml for ethambutol (EMB) and 2 µg/ml for streptomycin (STM). The interpretation of resistance was based on the standard criteria for resistance, i.e. 1% for all drugs (17).

RD9 Deletion Analysis
Region of difference 9 (RD9) deletion analyses was performed on heat-killed cells to confirm the presence or absence of RD9 for species identification of M. tuberculosis from the other members of MTBC as previously described (18). It uses three primers (RD9flankF, RD9intR and RD9flankR) for PCR reaction and the PCR amplification product was run by electrophoresis in 1.5% agarose gel and the results were interpreted as M.

Spoligotyping
Isolates that were positive for M. tuberculosis by RD9 PCR were further characterized by spoligotyping following the procedure described earlier (10). In brief, the direct repeat (DR) region of the isolate was amplified by PCR using oligonucleotide primers (DRa and DRb) derived from the DR sequence. Individual spoligotyping patterns were compared with the recent International Spoligotyping Database (SITVITWEB). Spoligotyping International Types (SIT) and sub-lineages (clades) were assigned according to signatures provided in SITVITWEB data base (http://www.pasteur-guadeloupe.fr:8081/SITVIT2/). An isolate was defined as a shared type if the same spoligotype was found in the database. If no matching spoligotype was found in the database, the isolate was defined as orphan (new).
Lineage types were assigned using a protocol for classification and analysis of MTBC (19).

Definitions
Newly diagnosed patients with pulmonary TB refer to patients who had never been previously treated for TB or have only taken anti-TB drugs for less than 1 month. M. tuberculosis isolates (two or more) that share the same genotype based on spoligotyping were considered clustered.

Data Management
All data were double entered into an online REDCap database (20) and analyzed using STATA software.

Ethical Consideration
The study was approved by the Institutional Review Boards of Addis Ababa University and the Armauer Hansen Research Institute (AHRI) as well as the Ethiopian National Ethics Review committee. Study permission was also obtained from the Oromia Regional Health Bureau, Western Arisi zone Health Department, Southern Regional Health Bureau and Sidama Zone Health Department.

Mapping of TB lineage and strain clusters
The spatial distribution of lineages identified in the study is presented in Fig. 3.
Mapping of the geographic location of clustered strains showed that the distribution of clustered strains varies within districts in the study area, and the highest proportions of clustered strains were observed in Wendo Genet district of Sidama Zone with SIT 149 (6.5%), SIT 699 (3.0%), and SIT 25 (2.6%). Shashemene town also had a higher proportion of clustered strains such as SIT 149 (4.4%), SIT 53 (4.8%) than other districts. Overall, clustered strains showed a varying distribution across districts and the districts such as Wondo Genet had all types of TB clusters. (Fig. 4).
Drug susceptibility profile  (Table 3). Table 3 Resistance to first-line anti-TB drugs and to streptomycin In the current study, the majority of the isolates (82%) belonged to the Euro-American lineage (L4) followed by East-Africa-Indian (L3), 14% and the Ethiopian lineage (L7), 4%. A recent study in southern Ethiopia (which was geographically close to our study) reported that 84% of the isolates were L4 and 3% of them were L3 (21). Studies from other parts of the country reported variable proportion of lineage types in different geographic areas of Ethiopia (12,22,23). Overall, L4 is more widely distributed and more predominant than all other lineages combined (24,25). On the other hand, a higher proportion of L3 (25-35%) was reported in northern Ethiopia than elsewhere (12,22). In general, it was noted that the geographic distribution and proportion of lineages varied across the country. The wider implication of this on the dynamics of the transmission of TB and drug resistance in the respective geographic localities is an area that has yet to be investigated well.
Lineage 7 accounted for 4% in the current study. One case of Lineage 7 was recently reported from the southern part of Ethiopia (21) and 6 cases (2%) from the southwest (26). Lineage 7 was first reported from Woldia area of Amhara region, Ethiopia with 13% of prevalence rate (12). Other studies from Amhara region (27,28) have reported prevalence rates of 10% and 16%. So far, lineage 7 has been prominently reported from the northern part of Ethiopia. The additional report of lineage 7 in the current study suggests its broader occurrence, including in the southern parts of the country.
Considering the pre-modern split of this lineage in the phylogenetic tree of M. tuberculosis and its localization to Ethiopia only, further investigations into its epidemiology would be of much interest.
In the current study, SIT 149 was the most common spoligotype (21%) circulating in the study area. Previous studies in Ethiopia have indicated that SIT149, also known as T3-ETH (29), ETH-3 and more recently as L4.2.ETH1 (25), is the most common spoligotype widely distributed in the country (24). It is also known to be more frequently associated with drug resistance than other spoligotype clusters (29). It is important that the distribution of this spoligotype is closely monitored and its drivers identified to better tailor control efforts.
Positioning of clustered strains using GIS mapping is helpful to describe the epidemiological links of Mtb strains in specific geographical localities (24). This information could be utilized to design targeted TB control measures. In the current study, geospatial analysis demonstrated variable distribution of strain clusters in the different districts of the study area suggesting areas affected with possible recent transmission.
Spoligotyping may correctly identify Mtb complex in to various lineages and sub-lineages, however it is known to overestimate clustering of isolates due to its inferior discriminatory power compared to other genotyping techniques (10,30,31). In general, clustering of strains is a marker for recent transmission and can also serve to evaluate the performance of the TB control program (32) and help determine where to target interventions.
In the current study, 14% of the newly diagnosed TB patients were resistant to ≥ 1 first-line anti-TB drugs. In terms of the burden of the problem, this prevalence could be considered as significant. However, the result is lower than the prevalence rates reported in other parts of the country such as 23% (13) in Central Ethiopia and 23% in Eastern Ethiopia (6), but relatively higher than the prevalence rate of 11% reported in Northern Ethiopia (8) and 9% in Southern Ethiopia (33). Compared to a similar study conducted in this same study area in 2006 (34), the prevalence reported in this study (14%) is relatively lower than that of the previous one, 20%. The difference in the prevalence rates observed in different parts of the country could be due to differences in TB control program performance but also, more simply, due to study-related factors such as study design (population differences, methodology employed in the studies, sample size, study participant selection methods) or study periods. In addition to active case finding, it is essential for programs to build capacity for DST in order to implement recommended early detection and effective treatment of drug resistance to curb its spread (13).
INH monoresistance was 10.9% in the current study and it is comparable with reports from eastern Ethiopia, 9.5% (6), but lower than 13.2% from western Ethiopia (35) and higher than reports from central Ethiopia, 4.7% (13) and 2.3% in the previous report from the same study area (34). INH monoresistance is the first step towards anti-TB drug resistance and it is the common pathway for the development of MDR-TB (36 (37). However, as it was indicated by Bekele and his colleagues (29), the observed association between T3-ETH (SIT 149) and development of drug resistance may not necessarily indicate that these strains are more prone to be drug-resistant but could rather be a consequence of their high prevalence in the population. This idea is also supported in a recent review (38) which showed that the correlation between genotypes and TB drug resistance was still uncertain. Further analysis on SIT149 identified genotype SIT149: A, a potential MDR-TB clone circulating in the Ethiopian highlands probably contributing to the spread of MDR-TB in the area that warrants further attention (29).

Limitations
The study had certain limitations. First, as our study participants were only newly diagnosed TB cases, it was not possible to assess the magnitude of drug resistant TB in the previously treated TB cases and the strain types among these groups. This has limited us from assessing the overall burden of drug resistant TB in the study area. Secondly, study participants were all patients seeking treatment at health facilities. Findings from such a selected population may not indicate the true burden of the problem at community level.

Conclusion
The study identified a heterogeneous pool of M. tuberculosis strains with several clusters including lineage 7 strains circulating in southern Ethiopia. No MDR-TB case was detected in the study area; however, the high proportion of INH monoresistance is a significant risk for the potential emergence of MDR-TB in the study area as INH monoresistance is the initial step towards anti-TB drug resistance and a common pathway to the development of